Dietmar E. Breithaupt

Identification and Characterization of a Mammalian Enzyme Catalyzing the Asymmetric Oxidative Cleavage of Provitamin A

In vertebrates, symmetricversus asymmetric cleavage of β-carotene in the biosynthesis of vitamin A and its derivatives has been controversially discussed. Recently we have been able to identify a cDNA encoding a metazoan β,β-carotene-15,15′-dioxygenase from the fruit flyDrosophila melanogaster. This enzyme catalyzes the key step in vitamin A biosynthesis, symmetrically cleaving β-carotene to give two molecules of retinal. Mutations in the corresponding gene are known to lead to a blind, vitamin A-deficient phenotype. Orthologs of this enzyme have very recently been found also in vertebrates and molecularly characterized. Here we report the identification of a cDNA from mouse encoding a second type of carotene dioxygenase catalyzing exclusively the asymmetric oxidative cleavage of β-carotene at the 9′,10′ double bond of β-carotene and resulting in the formation of β-apo-10′-carotenal and β-ionone, a substance known as a floral scent from roses, for example. Besides β-carotene, lycopene is also oxidatively cleaved by the enzyme. The deduced amino acid sequence shares significant sequence identity with the β,β-carotene-15,15′-dioxygenases, and the two enzyme types have several conserved motifs. To establish its occurrence in different vertebrates, we then attempted and succeeded in cloning cDNAs encoding this new type of carotene dioxygenase from human and zebrafish as well. As regards their possible role, the apocarotenals formed by this enzyme may be the precursors for the biosynthesis of retinoic acid or exert unknown physiological effects. Thus, in contrast toDrosophila, in vertebrates both symmetric and asymmetric cleavage pathways exist for carotenes, revealing a greater complexity of carotene metabolism.

Carotenol fatty acid esters: easy substrates for digestive enzymes?

To study the specificity of gastric lipases on carotenoid mono- and diesters, an enzymatic assay was applied. Digestions were carried out in phosphate buffer at pH 7.4 and 37 degrees C. As substrates we employed oleoresins from marigold (Tagetes erecta L.; lutein diesters), red paprika (Capsicum annuum L., mainly capsanthin diesters), papaya (Carica papaya L.; beta-cryptoxanthin esters), and loquat (Eriobotrya japonica Lindl.; beta-cryptoxanthin esters) as well as retinyl palmitate. These were reacted with porcine pancreatic lipase, porcine pancreatin, porcine cholesterol esterase, and human pancreatic lipase. As reference enzyme a yeast lipase from Candida rugosa was applied. A high turnover could be observed with porcine pancreatic lipase and porcine cholesterol esterase, indicating cholesterol esterase to be a plausible candidate for generation of free carotenoids in the gut. Human pancreatic lipase accepted only retinyl palmitate as substrate, carotenoid mono- and diesters were not hydrolyzed. The assay permits an approach for calculation of enzymatic activities towards carotenoid esters as substrates for the first time, which is based on the amount of enzyme formulation, present in the assay (U/mg solid). Furthermore, these studies provide deeper insight into carotenoid ester bioaccessibility.

Determination of folic acid by ion-pair RP-HPLC in vitamin-fortified fruit juices after solid-phase extraction

Abstract A sensitive and reliable method was developed for the determination of folic acid (FA) in vitamin-fortified fruit juices and fruit drinks. After solid-phase extraction clean-up with strong-anion-exchange material, FA was determined by ion-pair reversed phase high-performance liquid chromatography (RP-HPLC). Limits of detection (LOD) and quantitation (LOQ) were 0.04 and 0.06 mg/l, respectively. Average recoveries at two fortified levels (0.5 and 1.0 mg/l) were 97% in 0.1 M acetate buffer (pH 4.9) and ranged from 78 to 93% in spiked blackcurrant nectar, apple juice, and cherry nectar, with standard deviations less than 2.3%. The method was used to analyze nine commercial fruit juices and fruit drinks containing FA in the range of 0.30–1.40 mg/l. In two samples significantly less FA was found than specified.

Plasma response to a single dose of dietary β-cryptoxanthin esters from papaya ( Carica papaya L.) or non-esterified β-cryptoxanthin in adult human subjects: a comparative study

Many orange-coloured fruits contain beta-cryptoxanthin in its non-esterified as well as its esterified form. Information concerning the absorption of beta-cryptoxanthin, especially with regard to the metabolism of its fatty acid esters, is rather scarce. The present study assessed the plasma concentration reached after consumption of a single dose of native beta-cryptoxanthin esters from papaya (Carica papaya L.) or non-esterified beta-cryptoxanthin in equal total amounts. In a randomized, single-blind crossover study, twelve subjects were served a portion of yoghurt containing esterified or non-esterified beta-cryptoxanthin (1.3 mg absolute) together with a balanced breakfast. Between the two intervention days, there was a 2-week depletion period. After a fasting blood sample had been taken, futher samples were taken from the subjects at 3, 6, 9, 12 and 24 h. The concentration of non-esterified beta-cryptoxanthin in the whole plasma was determined by HPLC; beta-cryptoxanthin identification was confirmed by liquid chromatography-atmospheric pressure chemical ionization-MS analyses. Irrespective of the consumed diet, the plasma beta-cryptoxanthin concentrations increased significantly (P=0.05) and peaked after 6-12 h. The concentration curves, as well as the areas under the curves, were not distinguishable according to two-sided F and t tests (P=0.05). Standardization of beta-cryptoxanthin concentrations to plasma triacylglycerol and cholesterol had no impact on the results. Thus, the present study indicates comparable bioavailability of both non-esterified beta-cryptoxanthin and mixtures of beta-cryptoxanthin esters. The results support the existence of an effective enzymatic cleavage system accepting various beta-cryptoxanthin esters.

Enzymatic hydrolysis of carotenoid esters of marigold flowers (Tagetes erecta L.) and red paprika (Capsicum annuum L.) by commercial lipases and Pleurotus sapidus extracellular lipase

The carotenoids lutein and capsanthin, widely used as colorants, mainly occur as esters of fatty acids in their producer plants marigold and paprika. The enzymatic hydrolysis of these esters is reported here. Out of 26 commercial lipases, fastest ester hydrolysis under conditions was obtained using an enzyme from Candida antarctica (69% release of capsanthin, 44% of lutein from the respective oleoresins). Bile salts were essential for the activity of all commercial enzymes. A novel hydrolase was discovered in the mycelium-free supernatant of submerged cultures of the edible basidiomycete Pleurotus sapidus. The fungal enzyme cleaved several carotenoid esters almost completely and in the absence of bile salts.

Enzymatic Hydrolysis of Carotenoid Fatty Acid Esters of Red Pepper (Capsicum annuum L.) by a Lipase from Candida rugosa

Analyses of red pepper extracts which had been pretreated with lipase type VII (EC 3.1.1.3.) from Candida rugosa showed for the first time pepper carotenoid esters to be substrates of this enzyme. However, the extent of enzymatic hydrolysis depends on the respective carotenoid and was not quantitative compared to chemical saponification. After enzymatic cleavage, 67-89% of total capsanthin, 61-65% of total zeaxanthin, 70-81% of total β-cryptoxanthin and 70-86% of total violaxanthin were detected in free form. Nevertheless, the method described here offers the possibility to cleave in part several carotenoid esters originating from red pepper quickly and under comparatively mild reaction conditions. Replacement of the generally performed alkaline hydrolysis by enzymatic cleavage allows the resulting product to be used in food industry as “natural” coloring agent e.g. to colour cheese and jellies.

An extracellular carboxylesterase from the basidiomycete Pleurotus sapidus hydrolyses xanthophyll esters

Abstract An extracellular enzyme capable of efficient hydrolysis of xanthophyll esters was purified from culture supernatants of the basidiomycete Pleurotus sapidus. Under native conditions, the enzyme exhibited a molecular mass of 430 kDa, and SDS-PAGE data suggested a composition of eight identical subunits. Biochemical characterisation of the purified protein showed an isoelectric point of 4.5, and ideal hydrolysis conditions were observed at pH 5.8 and 40°C. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. An 1861-bp cDNA containing an open reading frame of 1641 bp was cloned from a cDNA library that showed ca. 40% homology to Candida rugosa lipases. The P. sapidus carboxylesterase represents the first enzyme of the lipase/esterase family from a basidiomycetous fungus that has been characterised at the molecular level.

Plasma responses in human subjects after ingestions of multiple doses of natural α-cryptoxanthin: A pilot study

Xanthophylls have attracted a lot of interest since their health benefits were documented. Unfortunately, studying their intestinal absorption is often affected by high baseline levels present in the fasting plasma. As alpha-cryptoxanthin is rarely found in the traditional European diet, its concentration in human plasma is extremely low. A pilot human intervention study was designed using alpha-cryptoxanthin for the first time as a marker xanthophyll in a minimally formulated cellulose-based supplement. Alpha-cryptoxanthin was administered in gelatin soft-gel capsules in multiple doses of 156 microg/d to three male volunteers (age 27.3 (SD 4.7) years; BMI 21.6 (SD 0.3) kg/m(2)) for 16 d after a 2-week carotenoid depletion period. Fasting blood samples were taken before the intervention and after 3, 6, 9, 13 and 16 d. Plasma HPLC analyses allowed for determination of the concentration; liquid chromatography-MS in the single ion monitoring mode was used to confirm peak assignment. The concentrations of alpha-cryptoxanthin increased significantly after only 3 d of supplementation. The concentration-time plots showed a characteristic shape with a first maximum after day 6, a decline until day 9 and a gradual second rise until the end of the study. Standardisation of plasma alpha-cryptoxanthin concentrations to triacylglycerol or total cholesterol did not influence the characteristics. The maximum concentrations reached at the end of the intervention period ranged from 0.077 to 0.160 micromol/l. These results suggest a high intestinal absorption and an enrichment of alpha-cryptoxanthin in the plasma even from a minimally formulated cellulose-based supplement.

Photoinduced addition of the fungicide anilazine to cyclohexene and methyl oleate as model compounds of plant cuticle constituents.

Photoreactions, initiated by sunlight irradiation, between organochlorine pesticides and olefinic compounds of plant cuticles have been postulated. Concerning the formation of bound residues, which so far have not been detectable by common analytical techniques, photoaddition reactions are of main interest. In order to study the photochemical behavior of chlorinated fungicides, anilazine was irradiated in cyclohexene and methyl oleate as model compounds for olefinic plant cuticle constituents. Anilazine extensively reacted with the cis-double bond of both model compounds via radical mechanisms. In addition to a dechlorinated photoproduct several addition products were formed showing plausible reaction pathways for the formation of bound residues in plant cuticles. Photoproducts were isolated by preparative HPLC and analyzed by HPLC, MS, 1H-, and 13C-NMR.

Comparison of the absorption efficiency of α-and β-cryptoxanthin in female Wistar rats

Xanthophylls, such as lutein and zeaxanthin, have received increasing interest in recent years because of positive correlations between their consumption and the prevention of eye diseases. Numerous human intervention studies have been conducted with lutein to estimate the bioavailability from different formulations. The present study was designed to obtain basic data on the absorbance efficiency of the monohydroxylated counterparts of lutein and zeaxanthin: alpha- and beta-cryptoxanthin. A corn-oil-based diet comprising beta-cryptoxanthin from papaya puree and alpha-cryptoxanthin from green carrot leaves was fed to five female Wistar rats for 8 consecutive days at a rate of 17.3 nmol/d and 9.2 nmol/d, respectively. The identity of the xanthophylls in the supplement was ascertained by LC-(APCI)MS analyses, and xanthophylls present in liver and plasma samples were determined by HPLC/diode array detector (DAD). The beta-cryptoxanthin concentrations of rat livers in the treatment group were statistically distinguishable (P < 0.01) from those present in the livers of the control group that were fed a basic diet. Alpha-cryptoxanthin, the second xanthophyll present in the supplement, was not found in rat livers in the treatment group. Plasma samples were free of xanthophylls. This is the first report proving that beta-cryptoxanthin has a higher absorption efficiency than alpha-cryptoxanthin in rats, at least from a minimally processed oil-based xanthophyll supplement.